CORE Metadata, citation and similar papers at core.ac.uk Provided by Digital.CSIC Novel peptides derived from αs1-casein with opioid activity and mucin stimulatory effect on HT29-MTX cells S. Fernández-Tomé1, D. Martínez-Maqueda1, R. Girón2, C. Goicoechea2, B. Miralles1, I. Recio1 1Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM, CEI UAM+CSIC), Nicolás Cabrera, 9, 28049 Madrid, Spain. 2Farmacología y Nutrición, Facultad de Ciencias de la Salud, Universidad Rey Juan Carlos, Unidad Asociada al IQM y al CIAL del CSIC, Avda. Atenas, s/n, 28922 Alcorcón, Madrid, Spain 1 ABSTRACT 143 149 This study investigates the opioid effect of αs1-casein fragments related to AYFYPEL and 144YFYPEL149, which had previously shown mucin-stimulatory activity in human goblet cells. Peptides 144YFYPEL149 and 144YFYPE148 showed opioid agonistic activity in guinea pig ileum, and in mouse vas deferens but to a lower extent. Peptides were partly hydrolysed during the assay and several of the resulting fragments lost the N-terminal Tyr residue. Docking of peptides 144YFYPEL149 (active) and 144YFYP147 (inactive) into the active site of the opioid receptor model showed remarkable differences regarding the flexibility at the third intracellular loop of the receptor and the interaction with Pro at the peptide C-terminus that forced residues Arg148 and Glu166 from the receptor to move towards the interior of the binding pocket. The study on 144 149 human cells HT29-MTX has shown that αs1-casein YFYPEL is the minimum fragment able to stimulate MUC5AC expression. KEYWORDS: Opioid peptide; mu-opioid receptor; molecular dynamics; mucin; goblet cells HIGHLIGHTS: 144YFYPEL149 and 144YFYPE148 demonstrated opioid activity on guinea pig ileum 144YFYPEL149 and 144YFYP147 presented different behaviour against opioid receptor 144YFYPEL149 was the minimum fragment with stimulatory effect on MUC5AC expression 2 1. INTRODUCTION During food digestion, proteins render a large variety of different peptides. Some of these sequences are structurally similar to endogenous physiologically active peptides and therefore, these food-derived sequences can interact with the same receptors in the organism, exerting an agonist or antagonist activity. One of the best examples where this applies, are food-derived opioid peptides, i.e., exogenous opioid receptor ligands with agonistic activity. From milk, peptides derived from β-casein, referred as β-casomorphins, were the first food protein-derived opioid receptor ligands whose sequences were identified (Brantl, Teschemacher, Henschen, & Lottspeich, 1979). They have been found in in vivo digestion products from humans and minipigs (Barbé et al., 2014; Boutrou et al., 2013). The common structural characteristics of both, exogenous and endogenous opioid peptides, are the presence of a tyrosine residue at the N-terminus and the presence of other aromatic residue, phenylalanine or tyrosine, in the third or fourth position (Meisel, 1997). In case of the αs1- casein derived peptides or α-casein exorphins, the active sequence can contain an additional arginine residue at the N-terminus. Although caseins are source of many peptides showing agonist or antagonist action on different opioid receptors, opioid peptides from other protein sources have been also described, such as whey proteins (Antila et al., 1991) and hemoglobin (Zhao, Garreau, Sannier, & Piot, 1997) from animal sources and gluten, rice, or soy from plant proteins (Yoshikawa, 2015). Biological activities observed for these food-derived opioid sequences include analgesia and modulation of social behaviour, after parenteral or intracerebral administration to animals. Orally administered food-derived opioid peptides have demonstrated to influence postprandial metabolism by stimulating secretion of insulin and somatostatin, prolongation of gastrointestinal transit time, stimulation of food intake, and effects on the immune system, among others (for reviews regarding biological activity, see Meisel & FitzGerald, 2000; Rutherfurd-Markwick, 2012; Teschemacher, 2003; Teschemacher, Koch & Brantl, 1997). 3 Moreover, it was found that the opioid peptide β-casomorphin-7 (60YPFPGPI66) induced intestinal mucin release through a nervous pathway and opioid receptor activation in ex-vivo preparations of rat jejunum (Claustre et al., 2002; Trompette et al., 2003). In human (HT29- MTX) and rat (DHE) intestinal mucin-producing cells, this peptide increased secretion and expression of mucin, and these effects were prevented with the μ-opioid antagonist cyprodime (Zoghbi et al., 2006). Similar effects were reported for the μ-opioid ligands, α- lactorphin (Martínez-Maqueda et al., 2012) and β-lactorphin (Martínez-Maqueda, Miralles, Ramos, & Recio, 2013b). However, other β-casein-derived peptides whose structures do not fulfil the requirements of opioid ligands have also demonstrated regulation of mucin production in HT29-MTX cells and in animals, such as the peptide β-CN f(94-123) found in yogurts and the derived fragments (94-108) and (117-123) (Plaisancié et al., 2013; Plaisancié et al., 2015). Our group had shown the mucin secretory activity of various bovine αs1-casein-derived peptides with favourable structures to bind opioid receptors due to the presence of Tyr at the N-terminus or in the second position and other Tyr in the third or fourth position. From these peptides, fragments 143AYFYPEL149, 144YFYPEL149 and a casein hydrolysate containing both sequences produced an increased mucin secretion and MUC5AC gene expression in HT29-MTX cells (Martínez-Maqueda, Miralles, Cruz-Huerta, Recio, 2013a). Interestingly, these two peptides had been identified in in vivo gastric and duodenal human digests after milk ingestion (Chabance et al., 1998). However, despite the favourable structure of these sequences and their potential to interact with opioid receptors located at the intestinal tract, the opioid activity of these peptides has not been previously demonstrated. The objective of this work 143 149 was to investigate the opioid effect of the bovine αs1-casein fragment AYFYPEL , and four derived peptides comprising the core structure for opioid activity YFY in guinea pig ileum and mouse vas deferens preparations. A molecular docking of two peptides into the active site of the μ-opioid receptor was carried out to identify the key residues responsible for the affinity to 4 the receptor. In addition, a preliminary study of these sequences on MUC5AC gene overexpression in HT29-MTX cells is shown. 2. MATERIALS AND METHODS 2.1. Peptides 143 149 144 149 144 148 144 147 144 146 αs1-casein fragments AYFYPEL , YFYPEL , YFYPE , YFYP and YFY , and the β-casein fragment 60YPFPGPI66 were synthesized in house using conventional solid- phase FMOC synthesis with a 433A peptide synthesizer (Applied Biosystems, Warrington, UK). In the peptide purification protocol, the treatment with acetic acid was included to replace trifluoroacetic acid which affects the pH of the peptide solutions in the bioassays. Their purity (>90%) was verified in our laboratory by reverse phase high performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS). 2.2. Isolated preparations from guinea pig ileum and mouse vas deferens Female guinea-pigs weighing 300-450 g, and male CD-1 mice weighing 25-60 g were used. Myenteric plexus-longitudinal muscle strips (MP-LM) from guinea pig ileum, and the mouse vas deferens were isolated as described by Ambache (1954) and Hughes et al. (1975), respectively. Tissues were suspended in a 10 ml organ bath containing 5 ml of Krebs solution (NaCl 118, KCl 4.75, CaCl2 2.54, KH2PO4 1.19; MgSO4 1.2; NaHCO3 25; glucose 11mM). This solution was continuously gassed with 95% O2 and 5% CO2. Tissues were kept under 1 g (guinea pig ileum) or 0.5 g (mouse vas deferens) of resting tension, at 37 ºC and were electrically stimulated through two platinum ring electrodes. MP-LM strips were stimulated with rectangular pulses of 70 V, 0.1 ms duration and 0.3 Hz frequency, and mouse vas deferens with trains of 15 rectangular pulses of 70 V, 15 Hz and 2 ms duration each min. The isometric force was monitored by computer using a MacLab data recording and analysis system. In both 5 assays the interval between applications of increasing concentrations was optimized to obtain a stable signal, and it was 9 min for αs1-casein peptides, and 3 min when control opioid agonists were tested. To evaluate the opioid-agonistic activity of peptides in the guinea pig ileum, cumulative concentration-response curves with five doses in the range 6.1 × 10-8 – 1.0 × 10-5 M were constructed in a step by step manner as follows: after 15 min-stabilisation of MP- LM strips in organ bath, electrical stimulation was applied, and peptide´s effect on the electrically induced contractions was evaluated once the response reached a plateau. Morphine was used as µ-opioid agonist positive control. To corroborate that the inhibitory effect of the peptides was mediated through interaction with opioid receptors, one dose of naloxone (1.0 × 10-6 M, Sigma), an non-selective opioid antagonist, was added to the organ bath at the end of each experiment. In mouse vas deferens preparations, non-cumulative concentration-response curves with four doses in the range 5.5 × 10-7 – 1.0 × 10-5 M were tested. Once tissue stabilisation, the first peptide dose was added and evaluated. The tissue was then washed, and subsequent doses were applied. In this preparation, [D-Pen(2),D- Pen(5)]-enkephalin (DPDPE) was used as δ-opioid agonist, and one dose of naltrindole (1.0 × 10-9 - 1.0 × 10-7 M, Sigma), a δ-selective opioid antagonist, was added after the experiments to evaluate selective interaction with δ-opioid receptors. Results were expressed as % of inhibition, taking the mean amplitude of the last five contractions before the addition of the peptides as 100%. In guinea pig ileum, when the effect of peptidase inhibitors (captopril, amastatin and phosphoramidon 1.0 × 10-6 M, Sigma) was evaluated, they were added 5 min before the beginning of the peptide´s concentration-response curve (Akahori et al., 2008).